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Originally published In Press as doi:10.1074/jbc.M606885200 on November 3, 2006

J. Biol. Chem., Vol. 282, Issue 2, 1238-1248, January 12, 2007
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Characterization of the Interleukin (IL)-6 Inhibitor IL-6-RFP

FUSED RECEPTOR DOMAINS ACT AS HIGH AFFINITY CYTOKINE-BINDING PROTEINS*

Silke Metz{ddagger}, Monique Wiesinger{ddagger}, Michael Vogt{ddagger}, Heike Lauks§, Günther Schmalzing§, Peter C. Heinrich{ddagger}, and Gerhard Müller-Newen{ddagger}1

From the {ddagger}Institut für Biochemie, Pauwelsstrasse 30 and the §Institut für Molekulare Pharmakologie, Universitätsklinikum RWTH Aachen, Wendlingweg 2, 52074 Aachen, Germany

Received for publication, July 19, 2006 , and in revised form, October 17, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although fusion proteins of the extracellular parts of receptor subunits termed cytokine traps turned out to be promising cytokine inhibitors for anti-cytokine therapies, their mode of action has not been analyzed. We developed a fusion protein consisting of the ligand binding domains of the IL-6 receptor subunits IL-6R{alpha} and gp130 that acts as a highly potent IL-6 inhibitor. Gp130 is a shared cytokine receptor also used by the IL-6-related cytokines oncostatin M and leukemia inhibitory factor. In this study, we have shown that the IL-6 receptor fusion protein (IL-6-RFP) is a specific IL-6 inhibitor that does not block oncostatin M or leukemia inhibitory factor. We characterized the complex of IL-6-RFP and fluorescently labeled IL-6 (YFPIL-6) by blue native PAGE and gel filtration. A 2-fold molar excess of IL-6-RFP over IL-6 was sufficient to entirely bind IL-6 in a complex with IL-6-RFP. As shown by treatment with urea and binding competition experiments, the complex of IL-6 and IL-6-RFP is more stable than the complex of IL-6, soluble IL-6R{alpha}, and soluble gp130. By live cell imaging, we have demonstrated that YFP-IL-6 bound to the surface of cells expressing gp130-CFP is removed from the plasma membrane upon the addition of IL-6-RFP. The apparent molecular mass of the IL-6·IL-6-RFP complex determined by blue native PAGE and gel filtration suggests that IL-6 is trapped in a structure analogous to the native hexameric IL-6 receptor complex. Thus, fusion of the ligand binding domains of heteromeric receptors leads to highly specific cytokine inhibitors with superior activity compared with the separate soluble receptors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytokines are important mediators in the regulation of immune responses and inflammation. Dysregulated cytokine signaling leads to chronic inflammation and cancer. Therefore, pro-inflammatory cytokines, such as tumor necrosis factor (TNF)2 and interleukin-1 and -6, have been identified as promising therapeutic targets. First approaches to specifically block the action of pro-inflammatory cytokines have focused on the use of neutralizing antibodies against a specific cytokine or its receptor. Only recently, the value of soluble cytokine receptors as cytokine antagonists for the treatment of inflammatory diseases has been fully recognized.

Pro-inflammatory cytokines signal through receptor proteins consisting of an extracellular part, a single transmembrane region and a cytoplasmic domain. Ligand binding to the extracellular part of the receptor results in the activation of signal transduction cascades by the cytoplasmic domain. Soluble receptors consisting only of the extracellular part are potent inhibitors of cytokine activity. They bind the cytokine with the same specificity and affinity as the membrane-bound receptors without eliciting an intracellular signal. A dimeric form of the soluble TNF receptor is currently used for the treatment of inflammatory diseases caused by elevated TNF expression (1).

Most cytokines signal through heteromeric receptor complexes consisting of two or more different receptor subunits. In such a case, inhibition of cytokine activity by soluble receptors is more challenging. Recently, we and others have shown that the appropriate fusion of different soluble receptor proteins results in highly potent antagonists (2, 3).

Interleukin-6 (IL-6) has been validated as a target for the treatment of several diseases, such as rheumatoid arthritis and the lymphoproliferative disorder known as Castleman disease (4, 5). IL-6 signals through two different receptor proteins. It first binds to an {alpha}-receptor subunit (IL-6R{alpha}). The low affinity complex of IL-6 and IL-6R{alpha} engages the signal transducing receptor subunit gp130 leading to a high affinity receptor complex and the initiation of the cytoplasmic signaling cascades. Upon ligand binding, Janus tyrosine kinases that are constitutively associated with gp130 become activated, resulting in tyrosine phosphorylation of the transcription factor STAT3 (signal transducer and activator of transcription 3). Activated STAT3 accumulates in the nucleus where it induces IL-6 target genes (6).

Soluble gp130 (sgp130) has a moderate antagonistic effect on IL-6 activity (7), whereas soluble IL-6R{alpha} (sIL-6R{alpha}) acts even agonistically (8). Together with IL-6, sIL-6R{alpha} can be regarded as a co-ligand required for the activation of gp130. We have shown that, in the presence of sgp130, the agonistic activity of sIL-6R{alpha} is converted to an antagonistic activity (9). Thus, the combined application of sIL-6R{alpha} and sgp130 results in a strong inhibition of IL-6 activity.

The extracellular part of gp130 consists of a single Ig-like domain (D1) followed by five fibronectin type III-like domains (D2–D6) (10). Domains D2 and D3 form the cytokine binding module (CBM), which is the structural hallmark of the hematopoietic cytokine receptors (11). The extracellular part of IL-6R{alpha} also consists of an Ig-like domain (D1) followed by a CBM (D2 and D3) (12). By mutational analysis, it was shown that IL-6 contacts its receptors with three distinct binding sites (13). Site I binds the CBM of IL-6R{alpha} (14), and sites II and III interact with the CBM and D1 of gp130, respectively (15).

Recently, the structure of the soluble hexameric IL-6 receptor complex consisting of two molecules of each IL-6, sIL-6R{alpha} (D2–D3), and sgp130 (D1–D3) has been solved by x-ray crystallography (16). One IL-6 molecule contacts with its site II the CBM of one gp130, and with its site III, the Ig-like domain of a second gp130 molecule. The same is valid for the second IL-6 molecule leading to a highly symmetric complex.

Based on the structural features of the IL-6 receptor complex and the well characterized antagonistic activity of the combination of sIL-6R{alpha} and sgp130, we designed a fusion protein (inter-leukin-6 receptor fusion protein, IL-6-RFP) consisting of the ligand binding domains of gp130 (D1–D3) and IL-6R{alpha} (D2–D3) fused with an appropriate peptide linker (see Fig. 7A) (3). We have shown that this fusion protein acts as a highly potent IL-6 inhibitor. The cytokine receptor gp130 is a shared receptor subunit that is used by other IL-6-type cytokines, such as inter-leukin-11 (IL-11), oncostatin M (OSM), and leukemia inhibitory factor (LIF) (17, 18). Although IL-6-RFP does not inhibit IL-11 (3), its activity toward LIF and OSM has not been tested yet.

Although the importance of fused cytokine receptors as future drugs has been recognized (19), their mode of action has not been analyzed in sufficient detail. Therefore, we investigated the specificity, stability, and stoichiometry of the complex formed by IL-6 and IL-6-RFP. We have shown that IL-6-RFP is a highly specific IL-6 inhibitor that does not interfere with the bioactivity of the related cytokines LIF and OSM. A new method for the analysis of protein-protein interactions based on blue native gel electrophoresis, fluorescent fusion proteins, and fluorescence scanning is presented. We have shown that a 2-fold molar excess of IL-6-RFP over IL-6 is sufficient to completely trap IL-6 in a binary IL-6·IL-6-RFP complex. The complex of IL-6·IL-6-RFP is more stable than the complex of IL-6 with the separate soluble receptors sIL-6R{alpha} and sgp130. As shown in a live cell imaging experiment, YFP-IL-6, once bound to its cell surface receptors, is removed from the plasma membrane upon the addition of IL-6-RFP. Analysis of the stoichiometry by native gel electrophoresis and gel filtration suggests that the architecture of the IL-6·IL-6-RFP complex is analogous to the hexameric receptor complex identified by x-ray crystal-lography. Thus, IL-6-RFP is a promising IL-6 inhibitor for the treatment of diseases caused by dysregulated IL-6 expression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cytokines, Cytokine Receptors—Recombinant human IL-6 was expressed in Escherichia coli, refolded, and purified as described previously (20). The specific activity of IL-6 was measured by a B9 cell proliferation assay (21). OSM was purchased from PeproTech (Rocky Hill, NJ) and LIF from Chemicon International (Temecula, CA). sIL-6R{alpha} was expressed in insect cells as previously described (22). sgp130 was obtained from R & D Systems (Minneapolis, MN).

Cell Culture and Transfection of HepG2 Cells—The human hepatoma cell line HepG2 (purchased from the American Type Culture Collection, Manassas, VA) was grown in Dulbecco's modified Eagle's medium F-12 1:1 mix with GlutaMaxTMI (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Cytogen, Princeton, NJ). For starvation conditions, cells were cultured in pure, serum-free Dulbecco's modified Eagle's medium with 4500 mg/liter glucose, GlutamaxTMI, and pyruvate (Invitrogen). Plasmids were transiently transfected into HepG2 cells using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions.

Cell Culture and Transfection of COS-7 Cells—The simian monkey kidney cell line COS-7 (kindly provided by I. M. Kerr, Cancer Research UK, London, UK) was cultivated in Dulbecco's modified Eagle's medium with GlutaMaxTM (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum, 100 mg/liter streptomycin, and 100,000 units/liter penicillin (Cambrex BioScience, Verviers, Belgium). Cells were grown at 37 °C in a water-saturated atmosphere in 5% CO2. Plasmids were transiently transfected into COS-7 cells using LipofectamineTM 2000 (Invitrogen) according to the manufacturer's protocol.

Reporter Gene Assay—HepG2 cells were seeded onto 6-well plates (9.6 cm2/well) and transiently co-transfected with pGL3-{alpha}2M-Luc (construct with luciferase gene regulated by the {alpha}2 macroglobulin promoter) and pCRTM3 LacZ (galactosidase construct with a constitutively active promoter; Amersham Biosciences). Cells were starved in serum-free medium for 6 h and subsequently stimulated with 5 ng/ml IL-6 or the combination of IL-6 and IL-6-RFP for 16 h at the molar ratios indicated. To determine the specificity of IL-6-RFP, HepG2 cells were stimulated in parallel with 0.5 ng/ml OSM or a mixture of OSM and IL-6-RFP, with 5 ng/ml LIF, or a mixture of LIF and IL-6-RFP. Previous to stimulation, the corresponding cytokine and IL-6-RFP were incubated for 30 min at 37 °C to allow complex formation. Preparation of cellular lysates and luciferase measurements were carried out according to the instructions of the manufacturer (Promega). The luciferase activity values were normalized to the transfection efficiency, which was determined as beta-galactosidase activity. The experiments were carried out in triplicates, and the mean values and standard deviations were calculated.

Preparation of Cell Lysates, SDS-PAGE, Western Blotting, and Immunodetection—HepG2 cells were cultured on 6-well plates, starved overnight, and stimulated with the indicated cytokine or combination of cytokine and IL-6-RFP for 20 min. Subsequently, cells were lysed with radioimmune precipitation assay lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM NaF, 15% glycerol, 20 mM beta-glycerophosphate, 1 mM Na3VO4, 0.25 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 1 µg/ml leupeptin). The lysates were analyzed with SDS-PAGE, Western blotting, and immunodetection using an antibody directed against phosphotyrosine (705)-STAT3 (Cell Signaling Technology, Danvers, MA) or STAT3 (type H190, Santa Cruz Biotechnology, Santa Cruz, CA). Both antibodies were used in a 1:1,000 dilution in TBS-N (20 mM Tris-HCl, pH 7.6, 137 mM NaCl and 0.1% Nonidet P-40).

Purification and Expression of YFP-IL-6 in Insect Cells—High five (H5) insect cells (Invitrogen) were cultured at 27 °C in Sf-900II medium (Invitrogen) containing 2 µg/ml blasticidin. To stably transfect the cells with an expression plasmid coding for YFP-IL-6, the Cellfectin method (Invitrogen) was used. 72 h after seeding of the stably transfected cells, the supernatants were harvested, and subsequently cell debris was removed by centrifugation and sterile filtration. YFP-IL-6 was purified from cell supernatants by affinity chromatography using immobilized sIL-6R{alpha}. The eluates were supplemented with 0.02% bovine serum albumin (BSA) to increase protein stability and dialyzed overnight against phosphate-buffered saline (0.2 M NaCl, 2.5 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4). The concentration of YFP-IL-6 in the eluted fractions was determined by SDS-PAGE, Western blotting, enzyme-linked immunosorbent assay (ELISA) (23), and fluorescence spectroscopy.

Fluorescence Spectroscopy—Samples containing YFP-IL-6 were excited at a wavelength of 514 nm, whereas the light emission between 520 and 600 nm was detected. The emission maximum of YFP at 525 nm was used for the determination of the YFP-IL-6 concentration.

Expression of IL-6-RFP in Baculovirus-infected Insect Cells— IL-6-RFP was cloned as described previously (3). The linker that joins D1–D3 of human gp130 with D2–D3 of human IL-6R{alpha} consists of the flexible extracellular "stalk" region of IL-6R{alpha} (Ala323-Val362) and is therefore supposed to be of low antigenicity. An additional N-glycosylation site is introduced with the linker (Asn-Ala-Thr) (3). To produce IL-6-RFP using the baculovirus expression system, the instructions given by the manufacturer were followed. The DNA encoding IL-6-RFP was cut out from the vector pSVL-IL-6-RFP by XbaI and BamHI (Roche Applied Science) and inserted into the polyhedrin locus-based baculovirus transfer vector pVL1392 digested with the same enzymes. Sf21 insect cells were cultivated at 27 °C in Sf-900II medium with 10% fetal calf serum. Cells were co-transfected with 4 µg of pVL1392-IL-6-RFP plasmid and 0.5 µgof BaculoGoldTM DNA. Single virus clones were obtained from the cell supernatants by end point dilution. Several clones were screened for expression of IL-6-RFP in Sf21 cells by ELISA. The selected virus clone was then amplified by infecting Sf158 cells cultivated at 27 °C in serum-free Sf-900II medium. For protein expression, exponentially growing Sf158 cells were infected with the recombinant IL-6-RFP baculovirus. Three days after infection, the cell supernatant containing IL-6-RFP (~1 µg/ml) was harvested and cleaned from cells and cellular debris by centrifugation and sterile filtration.

Purification of IL-6-RFP by Affinity Chromatography—IL-6-RFP was purified from Sf158 supernatants by affinity chromatography with IL-6 immobilized to CNBr-Sepharose (Amer-sham Biosciences). After applying the cell supernatants to the column and washing with phosphate-buffered saline containing 0.05% Tween 20, proteins were eluted with 1 M acetic acid and subsequently neutralized by 2 M Tris-HCl (pH 9). Fractions collected during affinity chromatography were analyzed by SDS-PAGE, silver-staining, Western blotting, and immunodetection with antibodies directed against sIL-6R{alpha} or FLAG (Sigma). The eluates were supplemented with 0.02% BSA (200 µg/ml) and dialyzed overnight against phosphate-buffered saline. The final concentration of purified IL-6-RFP was ~10 µg/ml at a yield of ~70%. IL-6-RFP was stored for up to three years at -20 °C without major loss of activity.

Quantification of IL-6-RFP by ELISA—An ELISA for the quantification of FLAG-tagged IL-6-RFP was carried out as described previously (9). The ELISA plates were coated with 0.3 µg/ml FLAG monoclonal antibody (Sigma), and 50 ng/ml biotinylated monoclonal antibody B-T2 (Diaclone, Besançon, France) was used as the secondary antibody. The standard curve was obtained by 2-fold serial dilutions of sgp130-FLAG expressed in COS-7 cells and calibrated by sgp130 ELISA (9).

Blue Native PAGE and Detection of Fluorescence of YFP—To allow complex formation, YFP-IL-6 and IL-6-RFP (or YFPIL-6, sIL-6R{alpha}, and sgp130) were incubated for 30 min at room temperature at molar ratios as indicated. Complexes were separated by blue native PAGE (24, 25) using gradient polyacryl-amide gels (from 4 to 12 or 4 to 20% polyacrylamide). The cathode buffer was composed of 50 mM tricine, 15 mM Bistris, and 0.002% Coomassie Brilliant Blue® G 250 (Serva, Heidelberg, Germany), whereas the anode buffer contained 50 mM Bistris/HCl, pH 7. BSA was used as molecular weight marker protein. In addition, BSA was utilized as an internal marker protein, because YFP-IL-6 and IL-6-RFP were supplemented with BSA to increase protein stability. The fluorescence of YFP was detected with a Typhoon gel imager (Amersham Biosciences) using an excitation at 488 nm, whereas the emission was detected using a 500–540 nm band-pass filter. After detection, the gels were fixed and silver-stained.

Live Cell Imaging by Confocal Laser Scanning Microscopy— Confocal microscopy of living cells was carried out using a Zeiss LSM 510 Meta confocal laser-scanning microscope (Zeiss, Jena, Germany) equipped with an argon laser, a 63x/1.2 aperture water-corrected objective, an open cell cultivation chamber (Pecon, Erbach, Germany), and a CO2 incubation and heating unit (Zeiss). For live cell imaging, COS-7 cells were transfected with pSVL-gp130id-CFP coding for an internalization-deficient mutant of gp130 fused to CFP (26). Subsequently, cells were seeded onto 42-mm glass coverslips, and 48 h after transfection, coverslips were placed into the open cell cultivation chamber. The CO2 incubation and heating unit maintained a constant CO2 amount of 5% and a temperature of 37 °C. For recording of multiple fluorescence signals from one cell, the multitrack function of the LSM 510 Meta microscope was used. Cyan fluorescence was excited with {lambda} = 458 nm (5% transmission) and detected after a band-pass filter BP 480/20. Yellow fluorescence was excited with {lambda} = 514 nm (5% transmission) and a band-pass filter BP 530–600 was used. All images represent confocal slices of ~1 µm. Cells were co-stimulated with 240 ng/ml YFP-IL-6 and 3 µg/ml sIL-6R{alpha}, and CFP and YFP fluorescence was measured every 4 min over 1 h. In a second approach, a 10-fold molar excess of IL-6-RFP over YFP-IL-6 was added to the cells 21 min after co-stimulation.


Figure 1
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  		FIGURE 1.
Bioactivity and specificity of IL-6-RFP. A, HepG2 cells were transfected with a reporter gene plasmid coding for luciferase under the control of the IL-6-responsive rat {alpha}2M promoter. To investigate the concentration dependence of the inhibition of the IL-6-induced luciferase activity by IL-6-RFP, IL-6 and IL-6-RFP were incubated at the indicated molar ratios for 30 min at 37 °C to allow complex formation. Subsequently, HepG2 cells were stimulated for 16 h with the mixture of cytokine and inhibitor. Luciferase activity was measured in triplicates. B, HepG2 cells were transfected as described for A and stimulated with IL-6, OSM, or LIF. To investigate the specificity of the IL-6 inhibitor, IL-6, OSM, or LIF were preincubated with IL-6-RFP for 30 min at 37 °C at the molar ratios indicated. Subsequently, HepG2 cells were stimulated for 16 h with the mixture of cytokine and inhibitor, and the luciferase activity induced upon the {alpha}2M promoter activation was measured. C, HepG2 cells were transfected as described for A. IL-6 was incubated with a 10-fold molar excess of IL-6-RFP for 30 min at 37 °C. Afterward, HepG2 cells were stimulated for 16 h with the mixture of cytokine and inhibitor (procedure a). In procedure b, HepG2 cells were preincubated with IL-6-RFP for 30 min and then stimulated with IL-6 for 16 h, whereas in procedure c, IL-6 and IL-6-RFP were added simultaneously to the cells without preincubation. In procedures d and e, HepG2 cells were stimulated with IL-6 for 10 or 20 min, respectively, and subsequently treated with IL-6-RFP for 16 h. After that time, luciferase activity induced upon {alpha}2M promoter activation was measured. D, HepG2 cells were seeded onto 6-well plates and serum-starved overnight. Subsequently, IL-6 was incubated with a 5-fold molar surplus of IL-6-RFP at 37 °C for 30 min to allow complex formation. The cells were stimulated with IL-6 or the mixture of IL-6 and IL-6-RFP for 20 min (procedure a). In procedure c, IL-6 and IL-6-RFP were added simultaneously to the cells (without preincubation) for 20 min. In procedures d and e, HepG2 cells were stimulated with IL-6 for 10 or 20 min, respectively, and afterward treated with IL-6-RFP for 20 min. E, OSM and IL-6-RFP or LIF and IL-6-RFP were preincubated for 30 min at 37 °C at the molar ratios indicated. HepG2 cells were stimulated with OSM or LIF or a mixture of OSM and IL-6-RFP or LIF and IL-6-RFP for 20 min. After stimulation, HepG2 cells were lysed. The lysates were analyzed by SDS-PAGE, Western blot, and immunodetection with phospho-STAT3- or STAT3-specific antibodies.

 
Gel Filtration—Gel filtration was carried out at 4 °C with a Superdex 200 16/60 column (Amersham Biosciences) at a flow rate of 1 ml/min. During each run, the light absorption at 280 nm was measured. The column was calibrated with 9 mg of a gel filtration standard protein mixture containing thyroglobulin (670 kDa), {gamma}-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12 (1.35 kDa) (Bio-Rad). During gel filtration of YFP-IL-6 and the YFP-IL-6·IL-6-RFP complex, 3-ml fractions were collected.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
IL-6-RFP Inhibits Acute Phase Protein Gene Induction and STAT3 Activation by IL-6 but Not by the Related Cytokines OSM and LIF—Human hepatocellular carcinoma cells (HepG2) respond to IL-6, OSM, and LIF stimulation with the induction of acute phase protein genes (27) and are therefore well suited to test the specificity of IL-6-RFP. As shown in Fig. 1A, IL-6-RFP concentration-dependently inhibited the induction of the {alpha}2 macroglobulin ({alpha}2M) promoter by IL-6 in a reporter gene assay. A 2-fold molar excess of IL-6-RFP over IL-6 led to a reduction of the biological response of ~70%. A >10-fold molar excess suppressed gene induction to nearly basal levels. At concentrations of IL-6-RFP, which completely inhibited IL-6 activity, gene induction by OSM was not disturbed (Fig. 1B). At a 30-fold molar excess of IL-6-RFP over LIF, LIF activity was not affected. A moderate inhibition of LIF activity was observed at a 60-fold excess. Fig. 1C shows that IL-6-RFP acts on IL-6-mediated reporter gene induction not only after preincubation of the cytokine and the inhibitor (procedure a) but also when the inhibitor is given simultaneously (procedures b and c) or with a delay of 10 min (procedure d)or 20 min (procedure e). Reporter gene induction was measured 16 h after cytokine stimulation, whereas STAT3 tyrosine phosphorylation could be detected within minutes (Fig. 1D). STAT3 activation was inhibited when IL-6 and IL-6-RFP were preincubated for 30 min (procedure a) or added simultaneously (procedure c). In this short term assay, a delay of the action of IL-6-RFP was not tolerated (procedures d and e). IL-6-RFP did not interfere with STAT3 activation induced by LIF or OSM (Fig. 1E).


Figure 2
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  		FIGURE 2.
Blue native PAGE; YFP-IL-6 binds to IL-6-RFP with a higher affinity than to sIL-6R{alpha} or the combination of sIL-6R{alpha} and sgp130. To compare the concentrations of IL-6-RFP (A), sIL-6R{alpha} (B), or the combination of sIL-6R{alpha} and sgp130 (C) sufficient to bind to YFP-IL-6, YFP-IL-6 was incubated with a 2-, 0.4-, 0.08- or 0.016-fold molar ratio of IL-6-RFP for 30 min at room temperature. In a parallel approach, YFP-IL-6 was mixed with a 10-, 2-, 0.4-, 0.08-, or 0.016-fold molar ratio of sIL-6R{alpha} or a 10-, 2-, 0.4-, 0.08-, or 0.016-fold molar ratio of the combination of sIL-6R{alpha} and sgp130 and also incubated for 30 min at room temperature. The protein complexes were separated by blue native PAGE, and the fluorescent YFP-IL-6 was visualized by fluorescence detection. Subsequently, the polyacrylamide gels were fixed and silver-stained.

 
Analysis of the Interaction between IL-6 and IL-6-RFP by a Fluorescence Gel Shift Assay—Although the bioactivity of IL-6-RFP has been analyzed in detail (Fig. 1) (3), the complex formation of IL-6-RFP and IL-6 has not been studied. In a recent publication, we have shown that fusion of the yellow fluorescent protein (YFP) to the N terminus of IL-6 does not affect its bioactivity (26). The fluorescent cytokine YFP-IL-6 was expressed in insect cells and purified by affinity chromatography. For stabilization of diluted solutions of YFP-IL-6, BSA was added. Different amounts of YFP-IL-6/BSA were loaded onto a non-denaturing polyacrylamide gradient gel, and electrophoresis was performed under blue native PAGE conditions. When the wet gel was analyzed by a fluorescence scanner, YFP-IL-6 was readily detectable as two separate bands (Fig. 2A, upper panel, lanes 2 and 3).

Subsequently, the same gel was silver-stained (Fig. 2A, lower panel). At the higher concentration (120 ng), the upper YFP-IL-6 band is clearly visible (Fig. 2A, filled triangle, lane 2), whereas the lower one is only weakly stained (open triangle, lane 2). At the lower concentration (24 ng) (Fig. 2A, lane 3), YFP-IL-6 is hardly visible. Thus, the sensitivity of the fluorescence detection was similar or even exceeded the sensitivity of a silver stain. In lane 1, albumin alone was loaded onto the gel. The albumin monomer (65 kDa) is the strongest band, but the dimer (130 kDa), trimer (195 kDa), and tetramer (260 kDa) are also clearly detectable. These bands serve as molecular mass markers. The calculated molecular mass of YFPIL-6 is 46 kDa. Therefore, the band between the albumin dimer (130 kDa) and the albumin monomer (65 kDa) corresponds to a dimer of YFP-IL-6 (92 kDa), whereas the lower band represents the monomer (46 kDa).

The addition of IL-6-RFP to YFPIL-6 resulted in a marked shift of the fluorescent band, which indicates complex formation of YFP-IL-6 with IL-6-RFP (Fig. 2A, lanes 4–6). We often observed that the band shift was accompanied with a considerable increase in fluorescence intensity. We attribute this phenomenon to quenching or dequenching of the fluorescence, dependent on varying local concentrations of Coomassie, salts, and polyacrylamide in the gradient gel. A 2-fold molar excess of IL-6-RFP was sufficient to trap YFP-IL-6 completely (lane 4). Still at a 0.4- or 0.08-fold molar ratio of IL-6-RFP over YFP-IL-6, a complex was formed. To achieve a complex formation between sIL-6R{alpha} and YFP-IL-6, a 10-fold molar excess of sIL-6R{alpha} is required (Fig. 2B, lane 8). The complex already disappears at a 2-fold molar excess (Fig. 2B, lane 9, compare with lane 4). The combination of sIL-6R{alpha} and sgp130 (Fig. 2C) required a 10-fold molar excess to completely bind YFP-IL-6, whereas a 2-fold molar excess led to only a partial trapping of YFP-IL-6 (Fig. 2A, compare with lane 4). These results indicate that IL-6-RFP binds YFP-IL-6 more efficiently than its soluble receptors.


Figure 3
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  		FIGURE 3.
Blue native PAGE; analysis of the stability of the complex formed by IL-6-RFP and YFP-IL-6 and the complex of YFP-IL-6·sIL-6R{alpha}·sgp130 in the presence of urea. To allow complex formation, 24 ng of YFP-IL-6 were incubated with a 2-fold molar surplus of IL-6-RFP (74 ng) for 30 min at room temperature (A). In a parallel approach (B), YFP-IL-6 (24 ng), sIL-6R{alpha} (44 ng), and sgp130 (74 ng) were mixed at a molar ratio of 1:2:2 and incubated at room temperature for 30 min to enable complex formation. Thereafter, the indicated amounts of urea were added and incubated for another 10 min at room temperature. After incubation, the protein complexes were separated by blue native PAGE, and the fluorescent YFP-IL-6 was visualized by fluorescence detection. Subsequently, the polyacrylamide gel was fixed and silver-stained.

 
IL-6 Forms a More Stable Complex with IL-6-RFP than with Its Soluble Receptors—Three experiments were performed to assess the stability of the complexes formed by IL-6 and IL-6-RFP or IL-6 and its soluble receptors. In a first experiment, the complexes were incubated in the presence of urea at increasing concentrations. Although the complex of IL-6·IL-6-RFP is still detectable in the presence of 6 M urea (Fig. 3A, filled triangle), only a faint band of the IL-6·sIL-6R{alpha}·sgp130 complex is visible in the presence of 2 M urea (Fig. 3B, filled triangle), which disappears at a concentration of 4 M urea. Interestingly, with increasing concentrations of urea, the band of the YFP-IL-6 monomer becomes more intense (Fig. 3), suggesting that urea induces a dissociation of the YFP-IL-6 dimers into monomers.

In a second approach, the complexes formed by YFP-IL-6 and IL-6-RFP or YFP-IL-6 and its soluble receptors were incubated with varying amounts of unlabeled IL-6 for 2 h to determine the replacement of YFP-IL-6 by IL-6. A 10-fold molar excess of IL-6 over YFP-IL-6 was required to detect a replacement of YFP-IL-6 in the complex of YFP-IL-6 and IL-6-RFP (Fig. 4A). The fluorescence intensity of the complex decreased at a 10-fold molar excess of IL-6 over YFP-IL-6 (Fig. 4A, filled triangle), whereas the fluorescence of the YFP dimer became more intense (open triangle). In the case of the receptor complex formed by YFP-IL-6 and its soluble receptors, lower amounts of unlabeled IL-6 were sufficient to displace YFP-IL-6 (Fig. 4B). The fluorescence of the YFP-IL-6 dimer increased when IL-6 was added at a 0.1-fold molar ratio (Fig. 4B, open triangle).

In a complementary third approach, increasing amounts of sIL-6R{alpha} and sgp130 were added to the complex formed by YFPIL-6 and IL-6-RFP (Fig. 4C). Hardly any YFP-IL-6·sIL-6R{alpha}·sgp130 ternary complex was detectable after 2 h. Conversely, when IL-6-RFP was added to a preformed YFP-IL-6·sIL-6R{alpha}·sgp130 ternary complex, the complex of YFP-IL-6·IL-6-RFP was formed within 2 h at only a 0.1-fold molar concentration of IL-6-RFP over sIL-6R{alpha} and sgp130 (Fig. 4D). Thus, the cytokine is more efficiently trapped in complex with IL-6-RFP than in complex with the soluble receptors.

IL-6-RFP Removes YFP-IL-6 from the Cell Surface Receptor Complex—Next, we asked whether IL-6-RFP, as a result of its superior activity, would be able to remove IL-6 from its receptor on the plasma membrane. Therefore, we transfected COS-7 cells with an internalization-deficient (id) mutant of gp130 fused to CFP (gp130id-CFP (26)) and analyzed living cells by confocal laser scanning microscopy. As shown in Fig. 5A, besides endoplasmic reticulum and Golgi apparatus, the plasma membrane of transfected cells is clearly visible in the CFP channel resulting from surface expression of gp130id-CFP. After the addition of YFP-IL-6 and sIL-6R{alpha} (COS-7 cells lack endogenous IL-6R{alpha}), the fluorescent cytokine accumulated at the cell surface resulting in a membrane staining detected in the YFP channel. The intensities of YFP and CFP fluorescence in the regions of interest at the plasma membrane (Fig. 5A, red rectangles) are depicted in Fig. 5B. YFP-IL-6 binding to gp130id-CFP reached saturation within 50 min (Fig. 5B, left panel).

In a second experiment (Fig. 5A, right panel), a 10-fold molar excess of IL-6-RFP over YFP-IL-6 was added 21 min after YFPIL-6/sIL-6R{alpha} stimulation when YFP-IL-6 binding was near saturation. The addition of IL-6-RFP resulted in a decrease of cell surface staining of YFP-IL-6, as shown in the image of the YFP channel 49 min after stimulation. The corresponding fluorescence intensities of YFP and CFP in the indicated region of interest are presented in the right diagram of Fig. 5B. After the addition of IL-6-RFP, the YFP-IL-6 fluorescence decreased, whereas gp130id-CFP fluorescence remained constant. The decrease of the YFP/CFP ratio at the plasma membrane indicates that YFP-IL-6 is removed from the cell surface. Within 25 min, the IL-6-occupied receptors decreased by 50%. Thus, IL-6-RFP has the capacity to eliminate IL-6 from activated receptor complexes at the cell surface.

Determination of the Molecular Mass of the YFP-IL-6·IL-6-RFP Complex by Gel Filtration—IL-6-RFP was purified from insect cell supernatants by affinity chromatography on IL-6-Sepharose (Fig. 6A). In non-reducing SDS-PAGE, IL-6-RFP appears as a monomeric protein with an apparent molecular mass of ~80 kDa (Fig. 6A, lane 2). We used a calibrated gel filtration column to determine the molecular masses of YFPIL-6, IL-6-RFP, and the complex of YFP-IL-6 and IL-6-RFP. YFP-IL-6 was detected by fluorescence spectroscopic analysis of the collected fractions. IL-6-RFP was quantified by ELISA (3). YFP-IL-6 alone eluted in two peaks from the gel filtration column (Fig. 6B). According to the retention times, these peaks correspond to YFP-IL-6 monomers (47 kDa) and YFP-IL-6 dimers (94 kDa). This finding is in line with the two species detected by blue native PAGE.


Figure 4
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  		FIGURE 4.
Blue native PAGE; competition of IL-6 with YFP-IL-6 trapped by IL-6-RFP or by sIL-6R{alpha} and sgp130. A, YFP-IL-6 was incubated with a 2-fold molar surplus of IL-6-RFP for 30 min at room temperature to allow complex formation. In a parallel approach (B), YFP-IL-6 was mixed with a 2-fold molar surplus of sIL-6R{alpha} and sgp130. Subsequently, non-tagged IL-6 was added at increasing concentrations (0-, 0.1-, 1-, 10-, and 100-fold molar ratio over YFP-IL-6) and incubated for 2 h at room temperature. The protein complexes were separated by blue native PAGE, and the fluorescent YFP-IL-6 was visualized by fluorescence detection. The complexes YFP-IL-6·IL-6-RFP (C) and YFP-IL-6·sIL-6R{alpha}·sgp130 (D) were preformed by incubation at 37 °C for 30 min at molar ratios 1:2 (C) or 1:10:10 (D). Subsequently, the complex YFP-IL-6·IL-6-RFP was incubated with a 0-, 0.1-, 1-, and 10-fold molar excess of sIL-6R{alpha} and sgp130 over IL-6-RFP for 2 h (C), and the complex YFP-IL-6·sIL-6R{alpha}·sgp130 was incubated with a 0-, 0.1-, 1-fold molar ratio of IL-6-RFP over the soluble receptors for 2 h (D).

 
The elution profile of IL-6-RFP is shown in Fig. 6C. Interestingly, no monomeric IL-6-RFP could be detected. The major portion of IL-6-RFP is found in a peak corresponding to 170 kDa that most probably represents the IL-6-RFP dimer. Also, some higher molecular mass species were detected.

Pre-incubation of YFP-IL-6 with a 2-fold molar excess of IL-6-RFP resulted in a dramatic change in the elution patterns (Fig. 6D). The whole population of YFP-IL-6 eluted earlier from the column corresponding to a higher apparent molecular mass. This finding indicates that YFP-IL-6 is completely trapped in a complex with IL-6-RFP, confirming the results obtained by blue native PAGE. Relevant fractions were analyzed by ELISA to detect IL-6-RFP (Fig. 6D, blue line). All fractions that contained the shifted YFP-IL-6 also contained IL-6-RFP. In addition, a second peak of IL-6-RFP devoid of YFP-IL-6 appeared. This peak corresponds to the unliganded IL-6-RFP dimer.

The apparent molecular mass of the YFP-IL-6·IL-6-RFP complex derived from gel filtration (Fig. 6D) is 325 kDa. If the complex is built up according to the native hexameric receptor complex (consisting of two molecules each of IL-6, IL-6R{alpha}, and gp130 (16)), a tetrameric complex is expected, consisting of two molecules of YFP-IL-6 and two molecules of IL-6-RFP. The calculated molecular mass for such a complex is 264 kDa. The discrepancy between the observed and calculated values can be attributed to the bulky architecture of the IL-6 receptor complex (16) as discussed below.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
According to the x-ray structure of the soluble IL-6 receptor complex, IL-6 signals through a hexam-eric complex consisting of two molecules each of IL-6, IL-6R{alpha}, and gp130 (16). The soluble receptors sIL-6R{alpha} and sgp130 are present in human blood in considerable concentrations (50 ng/ml (28, 29) and 300 ng/ml (7, 9), respectively). We found that the pairwise action of these soluble receptors blocks systemic IL-6 responses (9). Based on this observation, we designed a fusion protein composed of the ligand binding domains of IL-6R{alpha} (Fig. 7A, D2 and D3) and gp130 (D1–D3), which turned out to be a highly potent inhibitor of IL-6 (3). Such novel cytokine inhibitors are promising candidates for future drugs against chronic inflammation and autoimmune diseases (19). Therefore, a characterization of their mode of action is of considerable importance.


Figure 5
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  		FIGURE 5.
Live cell imaging of removal of YFP-IL-6 from the surface of gp130id-CFP-transfected cells by IL-6-RFP. A, COS-7 cells were transfected with 4 µg of pSVL-gp130id-CFP, seeded onto coverslips, and investigated in an open cell cultivation chamber by confocal laser scanning microscopy. Cells were stimulated with 240 ng/ml YFP-IL-6 and 3 µg/ml sIL-6R{alpha}, and pictures were taken every 4 min over 1 h (left panel). In a second approach (right panel), a 10-fold molar excess of IL-6-RFP was added to the cells 21 min after stimulation. The region of interest used for quantitative evaluation in B is marked in red. The cells shown are representative of seven cells in each approach. B, quantitative evaluation of A using the membrane region of interest indicated in each picture. The curve showing the CFP fluorescence is marked by blue triangles, whereas the curve representing the YFP fluorescence is marked by orange squares.

 
Because gp130 is the shared signaling receptor subunit of the IL-6-type cytokines, specificity of IL-6-RFP is a critical issue. On human cells, OSM signals through a heterodimer of gp130 and the OSM-receptor or a heterodimer of gp130 and the LIF-receptor (LIFR) (30). Even a large excess of IL-6-RFP over OSM does not inhibit OSM activity (Fig. 1, B and E), indicating that the affinity of OSM to the gp130 moiety of IL-6-RFP is very low. LIF signals through a heterodimer of gp130 and the LIFR (30). At a 60-fold molar excess of IL-6-RFP over LIF, LIF activity is moderately affected. This cross-reactivity of IL-6-RFP can be explained by low affinity binding of LIF to domains D2 and D3 of gp130 (31). However, at concentrations of IL-6-RFP sufficient to trap IL-6 in a high-affinity complex, the fusion protein is a highly specific IL-6 inhibitor. The low activity of IL-6-RFP toward OSM and LIF is in line with the ineffective neutralization of these cytokines by the full-length soluble gp130 (D1–D6). A 300-fold molar excess of sgp130 over OSM resulted in only a marginal inhibition of OSM bio-activity (7).

Interestingly, in a long term reporter gene assay that mimics IL-6 bioactivity, a short delay of IL-6-RFP application is well tolerated (Fig. 1C). To inhibit the early activation of STAT3 in response to IL-6, however, IL-6-RFP has to be added at least simultaneously (Fig. 1D). These results show that a short initial pulse of STAT3 activation is not sufficient for a robust gene induction but that the cytokine has to act for a considerable period of time. This means that, for the treatment of a disease with IL-6-RFP, the inhibitor can block the biological activity of IL-6 even when applied after the cytokine release. This is further substantiated by the results of our live cell imaging experiment. IL-6 bound to the cell surface is removed from the receptor complex upon the addition of IL-6-RFP (Fig. 5).

The shift of GFP fusion proteins in native gels can be used to detect protein-protein interactions (32). We combined the application of blue native gel electrophoresis, fluorescent fusion proteins, and advanced fluorescence gel imaging to analyze the interaction of IL-6-RFP with IL-6. YFPIL-6 was generated by fusion of the YFP to the N terminus of IL-6. The 28 N-terminal amino acids of IL-6 are not involved in receptor binding (33). Therefore, fusion of YFP to the N terminus of IL-6 does not affect IL-6 bioactivity (26). Indeed, the interaction of YFP-IL-6 with IL-6-RFP could be visualized by a dramatic shift of the fluorescent band (Fig. 2). A 2-fold molar excess of IL-6-RFP over YFP-IL-6 is sufficient to completely trap YFP-IL-6 in a high molecular mass complex. In these fluorescence gel shift assays, the separate soluble receptor proteins sIL-6R{alpha} and sgp130 are of lower activity, which is in line with their lower potential to inhibit IL-6 (3). The ranking of efficiency in trapping IL-6 is IL-6-RFP > sIL-6R{alpha}/sgp130 > sIL-6R{alpha} > sgp130. Sgp130 alone has no detectable affinity to IL-6 (not shown). The superior activity of IL-6-RFP is also reflected in the increased stability of the YFP-IL-6·IL-6-RFP complex toward denaturation by urea, the low rate of replacement of YFP-IL-6 by non-tagged IL-6, the resistance of the YFP-IL-6·IL-6-RFP complex toward an excess of the soluble receptor proteins (Figs. 3 and 4), and the elimination of receptor-bound YFP-IL-6 from receptor complexes at the plasma membrane by IL-6-RFP (Fig. 5).


Figure 6
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  		FIGURE 6.
Superdex gel filtration of YFP-IL-6 and the complex of YFP-IL-6·IL-6-RFP. A,25 µl of supernatant of baculovirus-infected insect cells containing ~25 ng of IL-6-RFP (lane 1) or 25 ng of purified IL-6-RFP in PBS (lane 2) were separated by SDS-PAGE under non-reducing conditions. Subsequently, the gel was silver-stained. M, marker proteins. B,57 µg of YFP-IL-6 were analyzed on a calibrated Superdex 200 16/60 column (molecular mass range 10–600 kDa) at a flow rate of 1 ml/min. 3-ml fractions were collected and analyzed by fluorescence spectroscopy measuring the YFP emission at 525 nm. C, IL-6-RFP (17.7 µg) was analyzed by gel filtration on the same column. 3-ml fractions were analyzed by ELISA. D, to enable complex formation, YFPIL-6 (5.7µg) was incubated with IL-6-RFP in a 2-fold molar surplus (17.7µg) for 30 min at room temperature. The complex was analyzed by gel filtration. The 3-ml fractions were analyzed by fluorescence spectroscopy to detect YFP-IL-6 and by an ELISA to determine the concentrations of IL-6-RFP. All molecular masses were calculated using a regression curve based on the separation of a known protein standard mixture.

 


Figure 7
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  		FIGURE 7.
Proposed assembly of the IL-6·IL-6-RFP complex in analogy to the hexameric IL-6 receptor complex. Individual domains of gp130 and IL-6R{alpha} are shown in blue and green, respectively. Black bars mark the conserved WSXWS motif in D3 domains, and black lines depict conserved disulfide bonds in D2 domains. The linker is shown as a black line. Interactions are designated as proposed by Boulanger et al. (16). Monomeric IL-6-RFP (A) forms a dimer (B) through site IIb and IIIb interactions. For clarity, in the complex of IL-6-RFP and IL-6 (C), only five of the ten interactions are marked. In the complex, each of the marked interactions occurs twice.

 
In blue native PAGE, the mobility of the complex of YFP-IL-6 and IL-6-RFP is lower than the mobility of the albumin tetramer (260 kDa) (Fig. 2A). From a plot of the logarithm of molecular masses of the BSA multimers versus the relative migration distances (Rf, not shown), we calculated an apparent molecular mass of ~290 kDa for the complex of YFP-IL-6 and IL-6-RFP. We performed gel filtration with a calibrated column (Fig. 6) to corroborate the molecular masses determined by blue native PAGE. Gel filtration confirmed the observations made by blue native PAGE. We found that preparations of YFPIL-6 consist of a monomeric and a dimeric species (Fig. 6B). Dimerization of YFP-IL-6 is not caused by the YFP moiety, because YFP appears as a monomer in native PAGE (not shown). Thus, dimerization of the fusion protein is attributed to the known property of recombinant IL-6 to form dimers. The metastable IL-6 dimer has been described to dissociate in the presence of urea with a midpoint of dissociation at 3 M urea (34). The behavior of YFP-IL-6 in our gel shift assays is in agreement with the biophysical investigations on the IL-6 dimer. The mobility of the IL-6·IL-6-RFP complex does not change under conditions that lead to dissociation of the YFP-IL-6 dimer (Fig. 3), indicating that IL-6-RFP is largely complexed with monomeric YFP-IL-6. This observation is in line with the fact that IL-6 dimerizes by interactions of the gp130 binding sites of two IL-6 molecules (34). These sites are occupied upon binding of IL-6 to IL-6-RFP (Fig. 7C).

Unexpectedly, unliganded IL-6-RFP elutes from the gel filtration column with a retention time corresponding to a dimeric protein. Sgp130 as well as sIL-6R{alpha} are monomeric proteins that interact in the presence of IL-6 (35). However, in the x-ray structure of the hexameric IL-6 receptor complex (16) four contacts between IL-6R{alpha} and gp130 were identified. Possibly, in the dimeric IL-6-RFP, these contacts are partially stabilized by the covalent linkage of the corresponding IL-6R{alpha} and gp130{alpha} domains leading to the formation of an IL-6-RFP dimer in the absence of IL-6 (Fig. 7B).

If we assume a preformed IL-6-RFP dimer as depicted in Fig. 7B, IL-6 fits well into this assembly, leading to a complex of IL-6 and IL-6-RFP built up in analogy to the hexameric IL-6 receptor complex (Fig. 7C). The apparent molecular masses determined by gel filtration (325 kDa) and blue native PAGE (290 kDa) are higher than the calculated molecular mass (264 kDa) of the (IL-6)2·(IL-6-RFP)2 complex. However, the complex depicted in Fig. 7C is not a highly compact structure but rather extended, comprising a real cavity between the two IL-6 molecules. This architecture might explain the increased apparent molecular mass. A similar deviation between calculated and apparent molecular masses has also been observed in analysis of the soluble IL-6 receptor complex by gel filtration and analytical ultracentrifugation (35).

Taken together our analysis by blue native PAGE and gel filtration suggests that the IL-6·IL-6-RFP complex is built up in close analogy to the soluble extracellular hexameric IL-6 receptor complex. The latter complex is held together by 10 protein-protein interactions (twice the sites of I, IIa, IIb, IIIa, and IIIb) (Fig. 7C). The superior stability of the IL-6·IL-6-RFP complex in comparison with a similar complex built up by IL-6, sIL-6R{alpha}, and sgp130 is caused by the stabilization of these interactions through a covalent linkage of the ligand binding domains of sIL-6R{alpha} and sgp130. Thus, a molecular basis is provided for the high specificity and superior affinity of IL-6-RFP in comparison to other IL-6 inhibitors. Therefore, the fusion of ligand binding domains of heteromeric cytokine receptors is a promising approach for novel anti-cytokine therapies.


   FOOTNOTES
 
* This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich SFB 542 and Graduiertenkolleg "Biointerface" GRK 1035) and the Fonds der Chemischen Industrie (Frankfurt am Main). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Institut für Biochemie, Universitätsklinikum RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany. Tel.: 49-241-80-88860; Fax: 49-241-80-2428; E-mail: mueller-newen{at}rwth-aachen.de.

2 The abbreviations used are: TNF, tumor necrosis factor; BSA, bovine serum albumin; CBM, cytokine binding module; CFP, cyan fluorescent protein; IL, interleukin; LIF, leukemia inhibitory factor; OSM, oncostatin M; RFP, receptor fusion protein; STAT, signal transducer and activator of transcription; YFP, yellow fluorescent protein; s, soluble; D, domain; R, receptor; ELISA, enzyme-linked immunosorbent assay; Bistris, 2-[bis(2-hydroxyethyl) amino]-2-(hydroxymethyl)propane-1,3-diol. Back


   ACKNOWLEDGMENTS
 
We thank Dr. John Wijdenes (Diaclone, Besançon, France) for the kind gift of the gp130 antibody B-T2. We thank Dr. Rene Krieg for his help with the fluorescence scanner and Andrea Küster for excellent technical assistance.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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S. Metz, G. Naeth, P. C. Heinrich, and G. Muller-Newen
Novel Inhibitors for Murine and Human Leukemia Inhibitory Factor Based on Fused Soluble Receptors
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